Figure 6.

ANAC046 and ANAC087 Redundantly Control Columella PCD.

(A) pUBQ:ToIM expression in the wild type, anac046-1, anac087-1, and anac087 anac046 was analyzed every 12 h (stages 1–3), with the visual onset of root cap shedding defined as stage 1. All three mutant lines show longer living shed columella cells compared with the wild type. EGFP signal is shown in green in the cytoplasm, and mRFP signal is shown in magenta in the vacuole. Vacuolar collapse indicating imminent cell death is visualized by merging of the green and magenta signals.

(B) Higher magnification of shed root cap cells from stage 2 and 3 shown in (A). Bars = 20 µm.

(C) and (D) Quantification of living, shed cells from the wild type and the anac046-1 mutant (C) and the wild type, anac087-1, and anac087 anac046 mutants (D) at three stages after shedding shows that columella cells of the mutants lived longer compared with the wild type. The columella cells of the anac087 anac046 double mutant also lived longer compared with the anac087-1 single mutant. Box plot whiskers show minimum and maximum values; + indicates mean of at least 10 roots. Poisson regression; ***P < 0.001 and ****P < 0.0001.

(E) At stage 0 (defined as 12 h before onset of shedding), anac087-1 and anac087 anac046 mutants with the pUBQ:ToIM show very elongated cells close to the root tips (indicated by arrowheads), which sometimes initiate shedding before the columella. eGFP is shown in green, and mRFP is shown in magenta. Bars in (A) and (E) = 50 µm.